High performance liquid chromatography (HPLC) is a very versatile technique used for the separation and purification of a wide variety of organic and inorganic substances. HPLC has been well described by Snyder and Kirkland in an "Introduction to Modern Liquid Chromatography," Snyder, L. R., and Kirkland, J. J., 2nd Ed., Wiley Interscience, N.Y. (1979). It differs from classic chromatographic methods in the size and nature of the packing materials. HPLC uses packing materials which have particle sizes int he range of 5 to 10 .mu.m and in which the particles are generally very porous. HPLC can be carried out in several modes; and they are, absorption of a compound of interest using polar supports (normal phase), absorption of the compound of interest with nonpolar supports (reversed phase), electrostatic interactions (ion exchange), ion pairing interaction (mixed absorption modes), size exclusion (separation based on molecular size), and affinity (biospecific binding).
The ideal support for HPLC would be a material that is extremely rigid, has a coefficient of cubic expansion of essentially zero under normal operating temperatures and has a suitable surface for further chemical derivatization. It would have a controlled geometry, that is, it would be spherical, have a monodispersed particle size distribution with controlled pore size. It would have appropriate surface properties for the intended chromatographic separation mode. And it would be inexpensive to manufacture.
The currently used HPLC packings may be broadly classified in two categories, one being organic based supports and the other being the inorganic-based supports. Silica gel and, in limited cases, alumina are preferred materials for inorganic-based supports. Silica gel may be used for chromatographic purposes without further surface modification, or the surface may be coated or chemically modified with organo silanes or other substances to offer the desired surface properties.
The advantage of an inorganic support, and particularly silica gel, is that it is a rigid material and can withstand pumping pressures of 10,000 to 15,000 psi and has a suitable surface for further chemical derivatization (silanol groups). It has controlled geometry and is relatively inexpensive to manufacture.
Marisi et al. describe in U.S. Pat. No. 2,408,986 a gel bead used as a catalyst. The beads are formed by increasing the pore size of inorganic gels by dispersion with finely divided combustible materials of 300 mesh size and smaller in gelable solutions. After gelation, the hydrogel is purified, dried, and the combustion material removed by oxidation to form pores larger than those present in the original gel structure.
The inorganic-based supports suffer from the disadvantages that when working with large biochemical molecules it is very difficult to eliminate the undesired interactions of the biomolecules with the modified silica surfaces. Also, in the pH range where most biochemical separations are performed the silica based supports tend to be unstable. Therefore, organic substances are widely used as HPLC supports in biochemical steudies. Organic supports can frequently be manufactured to have little or no undesirable interactions between the desired solute and the support. However, organic supports often have lower rigidity which results in lower maximum pumping pressures that could otherwise be used (.about.5,000 psi for the hard gels and.about.200 psi for the soft gels).
There are many examples in the art of processes for making various organic based supports. In U.S. Pat. No. 3,275,576 issued Sept. 27, 1966 to Flodkin et al. there is disclosed a process for preparing substitution products of hydrophilic high molecular weight copolymerizates of aliphatic hydroxyl group-containing substances with bifunctional organic substances. The products are obtained by reacting the hydroxyl groups of the copolymerizates with a monofunctional substance and are useful as cation-exchangers in separations. There is no disclosure by Flodkin et al. that these gels might have unusually high rigidity and therefore be able to withstand unusually high (for organic supports) pumping pressures.
In U.S. Pat. No. 3,652,540 issued Mar. 28, 1972, Determan et al. discloses a process for making ion exchangers which have as their base rounded particles of regenerated cellulose. These resins have improved flow properties over microcrystalline cellulose based materials but still suffer from resin compaction and reduced flow rates under higher pressures.
Kokaka et al. (U.S. Pat. No. 4,045,353 issued Aug. 30, 1977) disclose a process for producing chromatographic supports which have a microporous inorganic support and a radiation polymerized organic coating which has part of the polymer grafted to the surface of the inorganic support and a part of the polymer not bonded to the support. Kosaka et al. do not remove the inorganic core from the polymer prior to use as a chromatographic support.
U.S. Pat. No. 4,094,833 issued June 13, 1978 to Johansson et al. discloses a process for producing an improved dextran gel in particle form which has a much higher pore size and rigidity than previous dextran gels. This is accomplished by using a divinyl compound in the copolymerization reaction. Although this dextran has higher rigidity and improved flow properties over previous dextran gels it still is not suitable for use in HPLC applications.
Naofumi et al. [Journal of Chromatography 333, 107-114 (1984)] disclose the use of a polyvinyl alcohol gel to which benzamidine had been covalently linked for the separation of trypsin. Naofumi et al. achieve a flow rate of 1.7 mL per minute with a pressure drop n the column of less than 356 psi. The gel particles they used had a narrow-sized distribution of 9.+-.0.5 micrometers.
Kato et al. [Journal of Chromatography 333, 93-106 (1985)] report the use of a hydrophilic resin-based support for the reversed phase chromatography of proteins. Their support was developed by introducing phenol groups with an ether linkage into TSK gel G5000 pw. They report this material to be effective for the reverse phase chromatography of proteins using flow rates in the range of 0.5 to 1.5 mL per minute.
Hirata et al. [Journal of Chromatography, 396, 115-120 (1987)] discuss the performance of Asahipak GS columns (hydrophilic gels of vinyl alcohol copolymer) when exposed to various organic solvents. In all cases the use of organic solvents affects either swelling or shrinking in the gels. This is a very undesirable property for supports for use in HPLC.
Hjerten et al. [Journal of Chromatography 396, 101-113 (1987)] disclose a chromatographic support based on agarose crosslinked with divinyl sulphone. This support has enhanced rigidity compared to standard agarose but still can only withstanding pumping pressures up to 580 psi.
Porath [Journal of Chromatography 218, 241-259 36 (1981)] describes a process for preparing crosslinked agarose which has a higher rigidity than standard agarose. This process involves including particles that can be dissolved under conditions that do not disturb agarose (in a low agarose content gel). The gel is then contracted by washing with a suitable organic solvent followed by drying. The gel is then crosslinked in a solvent which does not re-swell the gel. The particles are then dissolved leaving a porous, crosslinked agarose. Porath does not discuss the pressures that these crosslinked agaroses can withstand without collapsing.
Most of the prior art appears to each producing porous microspheres from vinyl monomers using emulsion or suspension polymerization techniques such as:
______________________________________ Styrene - divinyl benzene Acrylonitrile-divinyl benzene (JOC 358 (1986) 129-136) Vinyl pyridine (JOC 354 (1986) 211-217) Vinyl alcohol (JOC 349 (1985) 323-329) Glycidyl methacrylate (JOC 376 (1986) 269-272) ______________________________________ JOC = Journal of Chromatography
These processes produce spherical polydisperse particle sizes and the mean particle size depends on the ratio of water to organic solvent, concentration and type of emulsifiers, rate of stirring, time, temperature, relative solubilities of reactants in aqueous and organic phases, and the nature and solubility of free radical generators. The matter is further complicated by trying to introduce pores of desired shape and size into the polymer matrix. This is usually accomplished by adding an inert substance (such as toluene) which is incorporated into the organic sphere but does not participate in the polymerization process. These processes are very tedious and the final results depend on a host of variables as noted above.
Another factor is the inherent linear nature of the vinyl copolymers in which crosslinking is introduced to add mechanical strength (usually divinyl benzene). These linear polymers, based on vinyl copolymers, tend to swell in organic solvents. This is a severe problem since the nature of the packed bed will change when exposed to different liquids causing changes in column permeability, back pressure, etc.
Monodispersed microspheres may be produced by swelling latex particles as described by Uglestadt et al. in PCT application NO 82/00052 published Apr. 28, 1983, Publication No. WO 83/01453. How to introduce pores in these materials is not taught. The process depends critically on the relative solubility of the monomers and polymers in water and organic phases and is sensitive to temperature, stirring, time and temperature.
Some of the prior art is based on crosslinked agarose. These materials are very sensitive to swelling in organic solvents, are very soft and unsuitable for HPLC.
In U.S. Pat. No. 4,010,242 issued Mar. 1, 1977 Iler et al. disclose a method of making uniform, porous oxide microspheres by forming a mixture of urea or melamine and formaldehyde in an aqueous sol containing colloidal oxide particles, copolymerizing the organic constituents and then burning out the organic constituent. Iler et al. do not discuss the possibility of dissolving the oxide particles after the copolymerization step to give a porous organic copolymer useful as a support in chromatographic separations.
The organic-based supports described to date generally suffer from a number of deficiencies as described above. In particular, They have insufficient rigidity and they will swell or shrink in organic solvents. Both of these properties cause the HPLC supports to collapse under high pumping pressures or gradients of varying organic solvent concentration. These problems reduce their chromatographic efficiency and render the supports useless for further chromatographic separations.